If the cDNA reaction isn't working and your RNA is good quality, it's probably bad enzyme. You could try checking your RNA on a gel to be sure there isn't any smearing that would indicate degradation. The nanodrop only measures nucleotide content, so it could give you numbers that look good, but the RNA is damaged. I've always had good success with BioRad's iScript cDNA synthesis if you want to try another kit.

...why are you speccing your cDNA?

Don't. Just...don't. It isn't necessary, and is in fact actively detrimental.

Your final cDNA synthesis reaction will include unincorporated nucleotides, unincorporated primers (oligodT or random hexamers/nonamers) and a variety of reducing agents and enzymes: it will tell you entirely misleading things about the DNA concentration, and will have a poor 260/280 because the reaction contains bucketloads of reverse transcriptase.

Assuming your RNA is of good quality (chiefly look for a high 260/230 ratio, ideally around 2.0) and gives you two sharp ribosomal bands on a gel, then your cDNA reaction should be assumed to faithfully replicate this in DNA form. So don't bother speccing the cDNA.

Second thing to consider: cDNA synthesis buffer usually contains all sorts of things that are good for cDNA synthesis, but detrimental for PCR, so you should dilute your final cDNA prep accordingly before using it in qPCR. You may need to determine the optimal dilution empirically, but I typically find a 1/10 dilution is sufficient to minimize downstream effects without over-dilution (which would make measuring rare transcripts harder).

So: your cDNA. How much are you using in qPCR, how are you assessing reproducibility, and what genes are you using to normalize?

Hi,

You should add a bit of specification about " My qPCR results are unreproducible ...". Generally, you should check the quality of each individual step (RNA, cDNA, qPCR). For the quality of the RNA, I suggest this great link:

https://www.researchgate.net/post/Can_anyone_help_me_with_gel_electrophoresis_of_total_RNA

There isn't much you can do to check the cDNA, as far as I know, other than to analyze the qPCR results. However, there is quite a number of things that can help to pinpoint your problem just by looking at your qPCR data. Maybe the starting point would be - do your curves look normal and are the technical controls working (the NTC does not amplify and technical replicates are OK)?

Hello Kaido and John,

Thank you both for your answers. My RNA always has very good ratios indicating good quality RNA, the only reason that I thought to nanodrop the cDNA was due to lack of reproducibility of the qPCR. I usually take 2000ng of RNA for each cDNA reaction and then dilute the cDNA 1:50 for my qPCR (and obviously further diluted for a standard curve).

My technical replicates are all very good and have been all given me an approximate CT value of 26, I have been using a verified GAPDH primer as to normalise. Curves all look normal. Controls show no amplification.

I have been using Bioline tetro cDNA synthesis kit; I have tried using 2 new kits to no avail so I do wonder if it is the RT enzyme. I have been recommended Invitrogen's Superscript VILO which is very pricey at £430- do either of you have experience using this reagent? I'll have a look at the BioRad's iScript cDNA kit too, thank you.

Your technical replicates are good, but your qPCR isn't reproducible?

These seem...mutually exclusive: could you clarify? If you're getting the same values for your GAPDH primers, then your cDNA should be fine, and the source of error lies with either your GOI primers, or your GOI itself.

What sort of Cq values are you getting for your genes of interest? Cq values of ~26 for GAPDH seems very low, especially with a reaction using 2ug of RNA. Using 800ng of RNA in a 10ul cDNA reaction, then diluting 1/10 and using 1ul per well (so ~8ng of cDNA per well) I generally find GAPDH to be in the 15-18 range. If you're getting values 10 cycles lower (corresponding to a ~1000-fold reduction in expression) for a gene as highly expressed as GAPDH, then this is a cause for concern:

1) perhaps your tissue/cells/whatever you're using for RNA don't express much GAPDH? If so, how sure are you GAPDH is an appropriate reference gene (and indeed, how sure are you of this anyway)?

2) perhaps you've over-diluted your cDNA (seems unlikely) or used too much RNA in the synthesis reaction: try using half that. Also, what sort of 260/230 ratios does your RNA have?